Transcription of mRNA in eukaryotes is performed by RNA polymerase II (RNAP II). The polymerase requires a large number of associated proteins that perform a variety of different functions during the transcriptional process. These factors bin to a unique C-terminal domain (CTD) of RNAP II that becomes extensively post-translationally modified. Temporal regulation of these binding events is achieved through control of the different CTD modifications, most notably phosphorylation of serine residues. The action of kinases on the CTD is critical for the various phospho-CTD associated proteins (PCAPs) to bind and carry out their activities at the appropriate stage of transcription. Aberrant kinase activity s associated with several pathological conditions where transcription is altered, including cancer and cardiac hypertrophy. Much as phosphorylation of the CTD is essential for RNAP II function, proline isomerization by the peptidyl prolyl isomerase Ess1 (Pin1 in humans) is essential for yeast viability. Ess1/Pin1 is known to act on the phospho-Ser-Pro bonds of the CTD, and was thought to restore the more favored transpeptide bond conformation (phosphorylation boosts the percentage of cis-Pro bonds). However, recent evidence has shown that the CTD phosphatase Ssu72 requires the cis conformation of the prolyl bond. This leads to the question of whether other binding partners similarly recognize cis-Pro over trans. While individual types of modifications have been extensively studied, it is clear that combinations of post-translational modifications on the CTD impact each other, affect binding of PCAPs, and need to be carefully studied. Obtaining this data is challenged by many factors, including the apparent lack of stringent specificity in antibodies used to detect distinct CTD modifications. This proposal will study the complex interplay of phosphorylation and proline isomerization and the resultant effects on binding and activities of CTD kinases using a library of CTD peptides that we will create. This will be accomplished through the use of a novel, high-throughput peptide array approach. Through the use of directed evolution of CTD binding protein scaffolds in conjunction with the library peptides and a yeast display system, we will develop protein segments with high specificity towards particular CTD modifications. Together, these experiments will advance our knowledge of the role of CTD modification in the transcriptional process and provide new, antibody-free reagents to examine the CTD during gene transcription.